Abstract [en]

Nijmegen breakage syndrome (NBS) is a rare genetic instability syndrome associated with a high incidence of lymphoid malignancies. The NBS1 protein has been implicated in telomere biology suggesting that cells from NBS patients might have deficient telomere maintenance capacity. In this study we characterized spontaneously immortalized T-cell lines derived from three NBS patients regarding growth characteristics, telomere biology, expression of cell-cycle regulators, and response to DNA damage to understand the role of NBS1 in the immortalization process. In all the NBS T-cell lines the acquisition of an immortal phenotype was associated with telomere length stabilization, high telomerase activity, and increased mRNA expression of the catalytic subunit of telomerase (hTERT), together with c-myc up-regulation. Our findings provide evidence that telomere length maintenance was intact in the T lymphocytes in the absence of a full-length NBS protein, presumably due to the presence of an alternatively transcribed NBS protein of 70 kDa. Normal protein expression patterns for pRb and p53 in all the immortal lines coincided with altered expression of some cell-cycle proteins as well as with an impaired G1/S arrest after gamma irradiation, despite a seemingly normal p53/p21 pathway. The here described, spontaneously immortalized NBS derived T-cell lines can be useful in future analysis of the biologic effects in the NBS.

Degerman, Sofie

Abstract [en]

Cellular immortalization is a major hallmark of cancer and is a multi-step process that requires numerous cell-type specific changes, including inactivation of control mechanisms and stabilization of telomere length. The telomeres at the chromosome ends are essential for genomic stability, and limit the growth potential of most cells. With each cell division, telomeres are shortened. Short telomeres may induce an irreversible growth arrest stage called senescence, or a growth crisis stage characterized by high genomic instability and cell death. Only very rarely do cells escape from crisis and become immortal, a stage that has been associated with the activation of the telomerase enzyme which can elongate and stabilize the telomeres.

The processes leading to senescence bypass, growth crisis escape and finally immortalization are only beginning to be elucidated. Most of our knowledge of the immortalization process is based on analyses of human fibroblast and epithelial cell cultures immortalized by genetic modification. In this thesis, spontaneously immortalized human T lymphocytes derived from patients with Nijmegen Breakage Syndrome and a healthy individual were used to identify critical events for senescence bypass and immortalization. Genetic analysis showed a clonal progression and non-random genetic changes including the amplification of chromosomal region 2p13-21 as an early event in the immortalization process. Telomere length gradually shortened at increasing population doublings and growth crisis was associated with critically short telomeres. The clone(s) that escaped growth crisis demonstrated a logarithmic growth curve, very short telomeres and, notably, no increase in telomerase activity or expression of the telomerase catalytic gene, hTERT. Instead, upregulation of telomerase activity and telomere length stabilization were late events in T lymphocyte immortalization. Escape from crisis was associated with downregulation of DNA damage response genes and altered expression of cell cycle regulators and genes controlling the cellular senescence program.

These data indicated that a number of layers of regulation are important in the process of immortalization and to provide further mechanistic detail, epigenetic analysis was carried out. Genome wide methylation array analysis identified early and step-wise methylation changes during the immortalization process. Interestingly, applying these findings to tumors of T cell origin revealed commonly methylated CpG sites in transformed cells. Deregulated gene expression of the polycomb complexes may have contributed to the epigenetic changes observed.

Taken together, our analysis of spontaneously immortalized T cell cultures identified several steps in the immortalization process including genetic, epigenetic, gene expression and telomere/telomerase regulatory events, contributing further insights to the complexity of cancer cell immortalization.